非洲猪瘟病毒RPA等温检测方法的建立  

Rapid and Sensitive Detection of African Swine Fever Virus by Recombinase Polymerase Amplification

作　　者：王建昌[1] 王金凤[1] 刘立兵[1] 孙晓霞[1] 袁万哲[2] 

机构地区：[1]河北出入境检验检疫局技术中心,河北石家庄050051 [2]河北农业大学动物医学院,河北保定071001

出　　处：《中国动物检疫》2016年第7期78-81,94,共5页China Animal Health Inspection

摘　　要：为建立一种简单、快速的非洲猪瘟病毒（ASFV）分子检测方法,基于ASFV P72基因保守序列,设计并合成引物,建立了一种ASFV重组酶聚合酶扩增（RPA）等温检测方法。结果表明,所建立的RPA方法在38℃水浴锅中恒温反应30 min,即可实现对目的片段的有效扩增;以包含ASFV P72基因的ORF质粒为模板,RPA的检测限达到10～2 copies,同世界动物卫生组织（OIE）推荐的实时荧光PCR方法检测限一致,但比OIE推荐方法的检测限高10倍;RPA仅特异性扩增ASFV P72基因,对FMDV、CSFV、PRRSV、PRV和PCV-2基因组c DNA或DNA没有扩增。本研究所建立的RPA方法操作简单、反应快速、检测成本低、结果确实可靠,为非洲猪瘟的一线防控提供了一种新的、可靠的技术支持。To develop an convenient and rapid molecular diagnostic method for the African swine fever virus（ASFV）, the recombinase polymerase amplification（RPA）was developed using primers specific for the conserved region of the ASFV P72 gent. The RPA reaction was performed successfully at 38℃ for 30 rain in a water bath. By using recombinant plasmid DNA carrying the P72 gene as template.The results showed that the detection limit of RPA was 102 copies of plasmid DNA, which was the same as the real-time PCR recommended by OIE, and it was 10 times more sensitive than it. RPA could not amplify templates of FMDV, CSFV, PRRSV, PRV or PCV-2, demonstrating high specificity.The ASFV RPA developed in the study is simple, rapid, cost-effective and reliable, which can be an novel and reliable tool for the control of ASF.

关 键 词：非洲猪瘟病毒 分子检测 等温扩增 聚合酶重组酶扩增 快速 

分 类 号：S852.651[农业科学—基础兽医学]